Multitarget-Based Cotreatment with Cilostazol and Celecoxib Synergistically Suppresses Collagen-Induced Arthritis in Mice by Enhancing Interleukin-10 T Expression
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International Immunopharmacology 73 (2019) 461–470 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Multitarget-based cotreatment with cilostazol and celecoxib synergistically suppresses collagen-induced arthritis in mice by enhancing interleukin-10 T expression So Youn Parka,c,1, Yi Sle Leea,c,1, Sang Yeob Leeb,1, Sung Won Leeb, Ki Whan Honga, ⁎ Chi Dae Kima,c,d, a Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan-si, Gyeongsangnam-do 50612, Republic of Korea b Department of Internal Medicine, College of Medicine, Dong-A University, Busan 49202, Republic of Korea c Department of Pharmacology, School of Medicine, Pusan National University, Yangsan-si, Gyeongsangnam-do 50612, Republic of Korea d Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Gyeongnam 50612, Republic of Korea ARTICLE INFO ABSTRACT Keywords: Cilostazol exerts potent anti-inflammatory effects and celecoxib, a COX-2 specific inhibitor, improves the un- Cilostazol satisfactory profile of NSAIDs. It was aimed to assess the anti-arthritic potential of celecoxib add-on for cilostazol Celecoxib therapy in collagen induced arthritis (CIA), and to elucidate the implication of interleukin (IL)-10 in the action of Rheumatoid arthritis cilostazol and celecoxib cotreatment. Cotreatment of RAW 264.7 cells with 10 μM cilostazol and 0.3 μM cel- RANKL ecoxib synergistically suppressed RANKL-induced increases in RANK mRNA and protein levels. When cultured in NFATc1 the presence of RANKL for 5 days, RANKL-stimulated expressions of osteoclastogenic genes (OSCAR, DC-STAMP, TRAP and cathepsin K mRNA) and the expression of RANK mRNA were markedly elevated. Furthermore, these gene expressions, including that of RANK, were significantly suppressed by cotreatment with cilostazol (10 μM) and celecoxib (0.3 μM). In addition, this co-treatment strongly down-regulated RANKL-induced NFATc1 protein and TRAP activity (key osteoclastogenic factors), and these down-regulations were significantly prevented by pre- treating cells with IL-10 neutralizing antibody. Furthermore, increased osteoclast formation and extensive re- sorption pit formation by bone marrow-derived monocytes obtained from C57BL/6 mice cultured in the pre- sence of M-CSF/RANKL were markedly suppressed by cilostazol and celecoxib cotreatment. Consequently, hindlimb paw thicknesses in DBA/1J CIA mice were significantly reduced by cilostazol (10 mg/kg/d) and cel- ecoxib (5 mg/kg/d) cotreatment. These results were accompanied by synergistic suppression of cartilage de- pletion and bone erosion and reductions in arthritis scores in the CIA mice. In conclusion, serum IL-10 levels in these mice were markedly increased by cilostazol and celecoxib cotreatment, whereas elevated serum IL-1β levels were markedly reduced. Cotreatment with low-dose cilostazol and celecoxib may ensure the synergistic anti-arthritic potential. 1. Introduction and severe pain [1]. Proinflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α secreted by activated T cells play crucial roles in the patho- Rheumatoid arthritis (RA) is a systemic autoimmune disease char- genesis of RA [2,3]. These cytokines stimulate synovial fibroblasts to acterized by systemic inflammation and multiple arthritis. In addition produce RANKL, which subsequently induces osteoclasts to cause de- to synovial lesions, bones at inflamed joints of RA patients are de- struction of cartilage and bone [4]. stroyed by activated osteoclasts, which leads to deformity, disability, RANKL, a member of the TNF family, plays important roles in Abbreviations: BMM, bone marrow-derived macrophage-like cells; COX, Cyclooxygenase; DC-STAMP, Dendritic cell-specific transmembrane protein; M-CSF, Macrophage colony-stimulating factor; NFATc1, Nuclear factor of activated T cells cytoplasmic1; OSCAR, osteoclast-associated receptor; RANK, receptor activator of nuclear factor kB; RANKL, Receptor activator of NF-κB ligand; RA, Rheumatoid arthritis; SFs, Synovial fibroblasts; TRAP, Tartrate-resistant acid phosphatase ⁎ Corresponding author at: Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan-si, Gyeongsangnam-do 50612, Republic of Korea. E-mail address: [email protected] (C.D. Kim). 1 These authors equally contributed to this work. https://doi.org/10.1016/j.intimp.2019.05.058 Received 19 March 2019; Received in revised form 16 May 2019; Accepted 28 May 2019 Available online 03 June 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved. S.Y. Park, et al. International Immunopharmacology 73 (2019) 461–470 osteoclast differentiation and activation [5,6]. Osteoclast precursor Table 1 cells derived from hematopoietic cells undergo differentiation to tar- Severity scoring system in arthritis. trate-resistant acid phosphatase (TRAP)-positive mononuclear cells in Clinical arthritis severity 0 Normal the presence of macrophage colony stimulating factor (M-CSF) and score 1 Slight erythema or swelling receptor activator of NF-κB ligand (RANKL), and then fuse to form 2 Distinct erythematous swelling multinucleated cells that resorb bone matrix [7,8]. Inflammatory cy- 3 Joint distortion 4 Ankylosis of the joint tokines and cyclooxygenase (COX)-2 that are released from rheumatoid Histological arthritis severity 0 Normal fi arthritis synovial broblasts (RASFs) are crucially involved in the de- score 1 Synovial inflammation - mild struction of articular bone and cartilage [9]. Furthermore, RANKL-in- Synovial lesion - mild alteration duced activation of RANK receptor on the surfaces of osteoclast pro- Cartilage destruction - mild Bone erosion -mild genitor cells leads to the activation of nuclear factor of activated T cells fl ff 2 Synovial in ammation - moderate cytoplasmic1 (NFATc1) that results in the di erentiation of osteoclast Synovial lesion - moderate alteration progenitor cells into pathological cells that finally fuse to form multi- Cartilage destruction - moderate nucleated osteoclasts [7,8]. Bone erosion - moderate When activated, RASFs play a key role in the pathogenesis of RA 3 Synovial inflammation - moderate Synovial lesion - severe destruction of the synovitis through pannus formation. The inflammatory cytokines, ma- synovia trix metalloproteinases (MMPs), and cyclooxygenase (COX)-2 are in- Cartilage destruction – severe destruction volved in the destruction of articular bone and cartilage [9,10]. As IL- with loss of cartilage 1β and TNF-α induce RASF proliferation and stimulating the produc- Bone erosion - severe destruction, with tion of osteoclasts in association with RANKL, the downregulations of disrupted joint architecture. IL-1β, COX-2, and NFATc1 is viewed a major target of pharmaceutical therapy. emulsified with an equal volume of Freund's complete adjuvant (Sigma) Cilostazol, an inhibitor of phosphodiesterase type III, suppresses intradermally at the tail base. 14 days after primary immunization, proliferation of synovial fibroblasts from RA patients by enhancing mice were boosted with bovine type II collagen supplemented with apoptosis with increased cytochrome c release and apoptosis-inducing incomplete Freund's adjuvant in the same manner 14 days later. 10 mg/ factor as well as increased caspase 3 activation via mediation of cAMP- kg cilostazol or 5 mg/kg celecoxib was administered intraperitoneally dependent protein kinase activation-coupled Nrf2-linked increased HO- beginning 3 days after the booster injection. Mice were euthanized on 1 expression [11]. More interestingly, when BV-2 microglia were ex- day 43, and knee joints were isolated. posed to LPS, nitrite levels significantly increased in conjunction with the expressions of iNOS and COX2, and these increased variables were significantly attenuated by cilostazol (3–30 μM) [12]. Interestingly, 2.3. Assessments of histopathological arthritis Park et al. [13] reported cilostazol markedly elevated IL-10 levels in RA macrophage culture media and significantly reduced LPS-induced in- Arthritis severities in individual limbs were assessed by evaluating creases in TNF-α and IL-1β production. Celecoxib (COX-2 specific in- erythema, swelling, and other changes. Clinical arthritis and histolo- hibitor) is a NSAID (non-steroidal anti-inflammatory drug) that selec- gical arthritis severities were scored, as previously described [17]. tively inhibits COX-2 and the production of PGE2 [14,15]. Celecoxib (Table 1). has also been reported to inhibit TNF-α-induced transcriptional activity κ and the DNA binding activity of NF- B[16], and this negative regula- 2.4. Quantification of cytokine immunostaining in joint tissues tion of NF-κB activation might importantly underlie its anti-in- fl ammatory activity. Tissue sections were obtained from paraffin blocks, rehydrated, and ff In the present study, we assessed the e ects of cilostazol and cel- incubated with anti-RANK and anti-TNFα antibodies. Immunoreaction ecoxib co-treatment on the up-regulation of serum IL-10 level, down- products were visualized using a broad-spectrum im- β regulation of serum IL-1 and inhibition of the osteoclastogenic genes munohistochemistry kit (Diaminobenzidine substrate kit, Vector NFATc1 and TRAP expressions, thereby resulted in synergistic decrease Laboratories, Inc., Burlingame, CA). in osteoclast formation and suppression of bone erosion in the CIA mouse under cotreatment with cilostazol and celecoxib. 2.5. Detection of osteoclasts and cartilage damage 2. Materials